We have demonstrated that the synthesis of the major complex-type oligosaccharide unit of glycoproteins is initiated by the en bloc transfer of a high molecular weight lipid-linked oligosaccharide (LLO) (Glc3Man9GlcNac2) to the nascent protein. This oligosaccharide is then processed by the removal of glucose and mannose residues and the addition of outer sugar residues to give rise to the mature oligosaccharide. We have also elucidated the complete structure of the LLO and the major processing intermediates and have proposed a scheme for the sequence of processing. We now propose to determine the sequence of mannose and glucose addition during the synthesis of the LLO. Cells will be labeled in vivo with radioactive sugar precursors and the biosynthetic LLO intermediates will be isolated and their structures determined. Preliminary results suggest that sugar addition is highly ordered. We will attempt to isolate and characterize the biosynthetic enzymes using LLOs of known structure as acceptors. We will also attempt to isolate minor species of LLOs which may serve special functions, such as being precursors of oligosaccharides of unusual structure. To facilitate these experiments, we will attempt to isolate new lectin-resistant mutants that have specific blocks in the synthesis of the major LLO and therefore must utilize minor pathways for glycosylation. We plan to isolate and characterize the glucosidases and mannosidases which carry out processing. We postulate that the conformation of the protein determines whether the protein-bound oligosaccharide is partially processed (i.e., to a high mannose-type oligosaccharide) or completely processed (i.e., to a complex-type oligosaccharide). To study this, we will incorporate amino acid analogs into the glycoproteins of enveloped viruses and immunoglobulins in an attempt to alter protein conformation and then determine whether processing of the oligosaccharide units of these molecules is altered. We will also compare the behavior of native glycoprotein to that of glycoprotein containing amino acid analogs when glycosylation of both has been inhibited with tunicamycin.